The adult human intestine contains up to 100 trillion microorganisms. These microflora play a central role in the maintenance and control of host homeostasis. For instance, intestinal microflora has a major protective role by displacing pathogens and enhancing barrier fortification, favoring development of the immune system and control of metabolic functions, yet, in genetically susceptible individuals, some components of the flora can contribute to the pathogenesis of various intestinal disorders including inflammatory bowel disease (IBD). In murine models of IBD, the presence of gut flora leads to the development of pathogenic immune responses that are held in check by Treg. In some cases, the role played by Treg cells is beneficial in limiting pathogenic effector responses, while in others, Treg control is excessive and leads to impaired development of protective responses. To maintain tonic immune responses, the GI tract needs to receive both regulatory and counterregulatory signalswhich implies that Treg cells have themselves to be controlled. Previous work showed that natural Treg can be controlled by Toll like receptor (TLR) signaling. Some interactions with TLR have been proposed to increase Treg suppressive capacity, while others have been shown to limit Treg function. The constant exposure of the gut immune system to a large number of microbial products in the gut provides a rational to consider the physiologic impact of TLR signaling on Treg homeostasis and function. Our aim is to investigate the mechanisms by which Treg are controlled in the GALT environment. In particular we are exploring the elements in the gut flora that favor or limit regulatory responses. TLR9 recognizes unmethylated cytosine phosphate guanosine (CpG) dinucleotides, which are abundant in prokaryotic DNA found in gut flora. Previous studies have shown that activation of DCs via TLR 9 can limit Treg suppressive function by enhancing the level of activation of effector T cells. We have recently shown that the presence of CpG at the time of Leishmania infection can limit accumulation of Treg in the infected dermis7. Previous work demonstrated an association of Crohns disease with a promoter polymorphism in the TLR9 gene in humans8. To determine if bacterial DNA could modulate Treg homeostasis in vivo, we evaluated the percentage of Treg in various compartments of TLR9&#8722;/&#8722;and WT mice. Nave TLR9&#8722;/&#8722;mice displayed a significantly higher percentage and absolute number of Treg compared with nave WT mice in all GALT compartments compared to WT mice but not in other tissues. In addition, the basal levels of IFN-&#947;and IL-17 produced by both CD4 and CD8+ T cells were reduced in the GALT of TLR9-/- mice . This modification of the GALT environment in TLR9-/- mice is associated with a poor response to oral infection with E.cuniculi and enhanced susceptibility to the parasite. In addition, the immune response to oral vaccination was also dramatically decreased. These impaired responses were the consequence of increased Treg frequency in TLR9-/- mice since in both cases, removal of Treg restored the capacity of the mice to mount an effector response. Thus, the increase of Treg in TLR9-/- mice limits both IFN-&#947;&#61472;and IL-17 responses in the GALT. We next assessed whether bacterial DNA from conventional gut flora has a direct adjuvant effect on the induction of immune responses in the gut. To reduce the impact of other innate signaling induced by gut flora, we treated mice with a cocktail of antibiotics in their drinking water. Mice were subsequently treated or not by gavage with bacterial DNA from mouse gut flora. Antibiotic treatment alone increased the percentage of Treg in the GALT but not in other peripheral tissues. Following antibiotic treatment, mice were vaccinated orally or infected with microsporidia and immune responses evaluated. Consistent with the increased percentage of Treg in the gut, antibiotic treatment alone reduced the capacity of mice to produce IFN &#947;and IL-17 following oral infection. Treatment of mice with bacterial DNA from conventional gut flora restored the response to a level comparable to control mice in all compartments. Similar data were obtained with CpG but not with LPS or other TLR ligands. On the other hand, reconstitution of mice with DNA extracted from probiotic DNA was not able to restore immune responses in the GI tract. Thus, our data demonstrate that gut flora DNA is sufficient for restriction of the regulatory response in the gut and maintenance of effector immune responses. Furthermore our data demonstrate that bacterial DNA from defined species are distinct in their capacity to control gut immune responses. To assess the mechanism underlying the increase of Treg and limitation of immune responses in the gut, we tested if bacterial DNA could limit the neo-generation of Treg. Our in vitro data clearly demonstrate that the addition of CpG or bacterial DNA prepared from gut flora but not other TLR ligands dramatically decreases the capacity of T cells to acquire Foxp3 when stimulated with LpDCs in vitro while enhancing their capacity to release IL-17. Our data support the hypothesis that the adjuvant role of bacterial DNA is associated with its capacity to inhibit peripheral conversion while favoring the emergence of effector T cells. Together, our data demonstrate that in the gut commensal bacterial DNA acts as a natural immunological adjuvant and critically controls the balance between Treg and effector T cell populations.